Method for diagnosing abnormality in tissue samples by combination of mass spectral and optical imaging

ABSTRACT

The invention generally relates to mass spectral analysis. In certain embodiments, methods of the invention involve analyzing a lipid containing sample using a mass spectrometry technique, in which the technique utilizes a liquid phase that does not destroy native tissue morphology during analysis.

RELATED APPLICATION

The present application claims the benefit of and priority to U.S.provisional patent application Ser. No. 61/487,363, filed May 18, 2011,the content of which is incorporated by reference herein in itsentirety.

GOVERNMENT INTEREST

This invention was made with government support under EB009459 awardedby National Institutes of Health. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The invention generally relates to mass spectral analysis.

BACKGROUND

Imaging mass spectrometry (MS) is currently in its translational phaseas a tool in medical histopathology. Many biological applications arebeing pursued due to its capability to provide comprehensive informationon the distribution of multiple endogenous and exogenous moleculeswithin animal tissues (van Hove ERA, Smith D F, & Heeren R M A (2010),J. Chromatogr. A 1217(25):3946-3954; Watrous J D, Alexandrov T, &Dorrestein P C (2011), Journal of Mass Spectrometry 46(2):209-222).Imaging MS has the capability of mapping drugs, metabolites, lipids,peptides and proteins in thin tissue sections with high specificity andwithout the need of fluorescent or radioactive labeling normally used inhistochemical protocols (Schwamborn K & Caprioli R M (2010), Mol. Oncol.4(6):529-538; and Chughtai K & Heeren R M A (2010), Chem. Rev.110(5):3237-3277).

Within the imaging MS techniques (Alberici R M, et al. (2010),Analytical and Bioanalytical Chemistry 398(1):265-294), ambientionization techniques such as desorption electrospray ionization massspectrometry (DESI-MS) have been rapidly emerging and have the advantageof being performed at atmospheric pressure without the need for samplepreparation (Ifa D R, Wu C P, Ouyang Z, & Cooks R G (2010), Analyst135(4):669-681). Other imaging techniques such as matrix assisted laserdesorption ionization (MALDI; Oppenheimer S R, Mi D M, Sanders M E, &Caprioli R M (2010), Journal of Proteome Research 9(5):2182-2190) andsecondary ion mass spectrometry (SIMS; Fletcher J S & Vickerman J C(2010), Analytical and Bioanalytical Chemistry 396(1):85-104) arecommonly performed under high-vacuum conditions and the former requirescareful sample preparation through the application of a matrix. Morerecently, much effort has been put towards advancing ambient imagingmass spectrometry within the biomedical field, especially in cancerdiagnostics (Dill A L, Eberlin L S, Ifa D R, & Cooks R G (2011),Chemical Communications 47(10):2741-2746). The prospect of improving theaccuracy of histopathological cancer evaluation by adding chemicalinformation to the morphological microscopic analysis, especiallyrelated to cancer diagnosis and grading represents an attainable andrelevant medical application. Nonetheless, technical challenges remainand validation studies are still needed to successfully mergemicroscopic and mass spectrometric information into routinehistopathology workflow.

As the ability of DESI-MS as a diagnostic tool is demonstrated in manystudies, this capability must be validated through extensive chemicaland microscopic examination of tissue sections and development ofclassification rules relating MS imaging molecular information totraditional pathology. The correlation between histology and DESI-MS hasthus far been performed by comparing the ion images obtained to thediagnosis from pathological evaluation of a serial hematoxylin and eosin(H&E) stained section (Masterson T A, et al. (2010) DistinctiveGlycerophospholipid Profiles of Human Seminoma and Adjacent NormalTissues by Desorption Electrospray Ionization Imaging Mass Spectrometry.J. Am. Soc. Mass. Spectrom. in press). Even though this strategy issufficient for optical image evaluation under routine microscopicpathology, workflow practicality and unambiguous correlation demand theuse of the same tissue section for morphological and MS imagingevaluation.

The first strategy reported for conducting histopathology and imaging MSanalysis on the same tissue section was the development of MALDI imagingcompatible dyes, which provide limited histological details (Chaurand P,et al. (2004), Analytical Chemistry 76(4):1145-1155). Tissue sectionstaining after MALDI imaging spectra acquisition and matrix removal isconsidered the most promising approach to pair MALDI imaging andhistological staining, a strategy named post-acquisition staining(Crecelius A C, et al. (2005), Journal of the American Society for MassSpectrometry 16(7):1093-1099). As an ambient imaging MS technique,DESI-MS frees the user from the need of a homogeneous matrix depositionon the sample.

A limitation preventing DESI-MS imaging compatibility withhistochemistry is that the most common DESI solvent systems,methanol/water and acetonitrile/water 1:1 (v/v), completely destroy thenative tissue morphology during analysis.

SUMMARY

The present invention provides new methodologies by which mass spectralanalysis of tissue (such as ambient tissue imaging by desorptionelectrospray ionization mass spectrometry) can be performed whilemorphology of the tissue section is kept intact or unmodified, allowingsubsequent analysis of the tissue by histochemistry or many othertechniques to be performed. This is a new methodology fornon-destructive, morphologically friendly tissue analysis by massspectrometry techniques, such as desorption electrospray ionization massspectrometry. Thus in certain aspects, the invention provides methodsfor analyzing tissue that involve analyzing a tissue sample using a massspectrometry technique, in which the technique utilizes a liquid phasethat does not destroy native tissue morphology during analysis. Incertain embodiments, analyzing involves imaging a tissue section.

In certain embodiments, methods of the invention allow extraction oflipid species from tissue during DESI-MS analysis while morphology ofthe tissue remains undisturbed, therefore allowing subsequent analysisto be performed on the same tissue section. Particularly, methods of theinvention allow high-quality 2D DESI-MS ion images to be directlycompared and even overlaid with the H&E stained tissue section, allowinga better correlation between the spatial distribution of the lipidspecies detected and the substructures of a subject's brain.Pathological evaluation of the tissue sections confirmed that nomorphological damage was caused to the tissue as a result of DESI-MSimaging when using appropriate solvents.

Importantly, methods of the invention allow for DESI-MS imaging of anytype of sample that includes lipids, for example, human or animaltissue, plant tissue, soil, industrial chemical mixtures, and cleaningmaterials. In certain embodiments, the sample is human tissue. The humantissue may be epithelium tissue, healthy or diseased, such as cancerousbladder, kidney and prostate tissue. In these embodiments, DESI-MSimaging may be performed on the tissue to obtain a molecular diagnosisand then the same tissue section can be used not only for H&E staining,but also for immunohistochemistry. These advancements allow DESI-MSimaging to be included in the tissue analysis clinical workflow. Theyalso allow more detailed diagnostic information to be obtained bycombining two orthogonal techniques, imaging MS and histologicalexamination.

In other aspects, the invention provides methods for imaging a lipidcontaining sample (e.g., a tissue sample) that involve imaging a lipidcontaining sample using a direct ambient ionization/sampling technique,in which the technique is performed in a manner that allows the sampleto be subjected to further analysis after imaging.

Another aspect of the invention provides analysis methods that involveobtaining a lipid containing sample, imaging the sample using a massspectrometry technique, in which the technique utilizes a liquid phasethat does not destroy native tissue morphology during analysis, andperforming a histochemistry analysis technique on the sample.

Another aspect of the invention provides methods for diagnosing cancerthat involve obtaining a lipid containing sample, imaging the sampleusing a mass spectrometry technique, in which the technique utilizes aliquid phase that does not destroy native tissue morphology duringanalysis, performing a histochemistry analysis technique on the sample,and diagnosing a cancer based results of the imaging and the performingsteps.

Any mass spectrometry technique known in the art may be used withmethods of the invention. Exemplary mass spectrometry techniques thatutilize ionization sources at atmospheric pressure for mass spectrometryinclude electrospray ionization (ESI; Fenn et al., Science, 246:64-71,1989; and Yamashita et al., J. Phys. Chem., 88:4451-4459, 1984);atmospheric pressure ionization (APCI; Carroll et al., Anal. Chem.47:2369-2373, 1975); and atmospheric pressure matrix assisted laserdesorption ionization (AP-MALDI; Laiko et al. Anal. Chem., 72:652-657,2000; and Tanaka et al. Rapid Commun. Mass Spectrom., 2:151-153, 1988).The content of each of these references in incorporated by referenceherein its entirety.

Exemplary mass spectrometry techniques that utilize direct ambientionization/sampling methods including desorption electrospray ionization(DESI; Takats et al., Science, 306:471-473, 2004 and U.S. Pat. No.7,335,897); direct analysis in real time (DART; Cody et al., Anal.Chem., 77:2297-2302, 2005); Atmospheric Pressure Dielectric BarrierDischarge Ionization (DBDI; Kogelschatz, Plasma Chemistry and PlasmaProcessing, 23:1-46, 2003, and PCT international publication number WO2009/102766), and electrospray-assisted laser desoption/ionization(ELDI; Shiea et al., J. Rapid Communications in Mass Spectrometry,19:3701-3704, 2005). The content of each of these references inincorporated by reference herein its entirety.

In certain embodiments, the mass spectrometry technique is desorptionelectrospray ionization (DESI). DESI is an ambient ionization methodthat allows the direct ionization of species from thin tissue sections(Takats et al., Science, 306:471-473, 2004 and Takats, U.S. Pat. No.7,335,897). DESI-MS imaging has been successfully used to diagnosemultiple types of human cancers based on their lipid profiles detecteddirectly from tissue (Eberlin L S, Ferreira C R, Dill A L, Ifa D R, &Cooks R G (2011) Desorption Electrospray Ionization Mass Spectrometryfor Lipid Characterization and Biological Tissue Imaging. Biochimica EtBiophysica Acta-Molecular And Cell Biology Of Lipids accepted).

Human bladder cancer and adjacent normal tissues were successfullydistinguished on the basis of multiple marker lipids (Cooks R G, et al.(2011), Faraday Discussions 149:247-267). Multivariate statisticalanalysis of the DESI-MS imaging data by means of principal componentanalysis and partial least squares discriminant analysis allowed asuccessful correlation between DESI-MS data and pathological evaluationin 88% of the cases analyzed (Dill A L, et al. (2011), Chemistry-aEuropean Journal 17(10):2897-2902). DESI-MS imaging was also applied forthe diagnosis of human cancers including; two types of kidney cancer(Dill A L, et al. (2010), Analytical and Bioanalytical Chemistry398(7-8):2969-2978); human prostate cancer (Eberlin LS, et al. (2010),Analytical Chemistry 82(9):3430-3434); and the grading of brain gliomas(WHO grade II, grade III and grade IV (glioblastoma; Eberlin LS, et al.(2010), Angewandte Chemie-International Edition 49(34):5953-5956). Inaddition to cancer diagnostics, DESI-MS imaging has been used tocharacterize tissues of other disease states, such as chemicallyprofiling and imaging of human arterial plaques with atherosclerosis(Manicke N E, et al. (2009), Analytical Chemistry 81(21):8702-8707). Inaddition to the possibility of supplementing the DESI-MS solvent withionization facilitator compounds (Jackson A U, Shum T, Sokol E, Dill A,& Cooks R G (2011), Analytical and Bioanalytical Chemistry399(1):367-376), a unique capability of DESI-MS is the possibility touse reactants in the solvent to facilitate the ionization (reactiveDESI) and detect important metabolic intermediates that can be difficultto ionize, such as cholesterol (Wu C P, Ifa D R, Manicke N E, & Cooks RG (2009), Analytical Chemistry 81(18):7618-7624).

Operated in an imaging mode, it uses a standard microprobe imagingprocedure, which in this case involves moving the probe spraycontinuously across the surface while recording mass spectra. See forexample, Wiseman et al. Nat. Protoc., 3:517, 2008, the content of whichis incorporated by reference herein its entirety. Each pixel yields amass spectrum, which can then be compiled to create an image showing thespatial distribution of a particular compound or compounds. Such animage allows one to visualize the differences in the distribution ofparticular compounds over the lipid containing sample (e.g., a tissuesection). If independent information on biological properties of thesample are available, then the MS spatial distribution can providechemical correlations with biological function or morphology. More over,the combination of the information from mass spectrometry andhistochemical imaging can be used to improve the quality of diagnosis.

In particular embodiments, the DESI ion source is a source configured asdescribed in Ifa et al. (Int. J. Mass Spec/rom. 259(8), 2007). Asoftware program allows the conversion of the XCalibur 2.0 mass spectrafiles (.raw) into a format compatible with the Biomap software(freeware, htto://www.maldo-msi.org). Spatially accurate images areassembled using the Biomap software.

Methods of the invention involve using a liquid phase that does notdestroy native tissue morphology. Any liquid phase that does not destroynative tissue morphology and is compatible with mass spectrometry may beused with methods of the invention. Exemplary liquid phases include DMF,ACN, and THF. In certain embodiments, the liquid phase is DMF. Incertain embodiments, the DMF is used in combination with anothercomponent, such as EtOH, H₂O, ACN, and a combination thereof. Otherexemplary liquid phases that do not destroy native tissue morphologyinclude ACN:EtOH, MeOH:CHCl₃, and ACN:CHCl₃.

In certain embodiments, methods of the invention involve performing ahistochemical analysis on the tissue sample after it has been subjectedto mass spectrometric analysis. Any histochemical analytical techniqueknown in the art may be performed on the tissue, and the performedtechnique will depend on the goal of the analysis. Exemplaryhistochemical analytical techniques include H&E staining orimmunohistochemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Chemical information and physical effect of (A) standard DESI-MSsolvent system MEOH:H₂O (1:1) and new solvent systems (B) DMF:EtOH (1:1)and (C) DMF:H₂O (1:1) on sequential 15 μm thick mouse brain tissuesections. DESI-MS mass spectra of similar regions of the gray brainmatter obtained with (A) MEOH:H₂O (1:1) and (B) DMF:EtOH (1:1) solventsystems show similar molecular information, while (C) DMF:H₂O (1:1)favored the ionization of smaller m/z molecules such as fatty acids andmetabolites. Insets (LHS) show an optical image of the DESI-MS imagingexperiment on mouse brain tissue. The physical damage to the tissue isstrikingly different between the standard solvent systems and the newmorphologically friendly solvent systems. Insets (RHS) show magnifiedbright-field optical images of the same region of the different mousebrain tissue sections which were first imaged by DESI-MS with thedifferent solvent systems and subsequently H&E stained.

FIG. 2. Repeated DESI-MS imaging analysis of a mouse brain tissuesection using DMF:EtOH as the solvent system. Mass spectra of graymatter region of a 15 μm thick mouse brain tissue section is shown forthe (A) 1^(st) and (A) 10^(th) DESI-MS analysis of the same tissuesection. A plot of the total intensity of the major ion m/z 834.4 (PS18:0/22:6) was obtained for the 15 μm thick mouse brain tissue and for a5 μm thick mouse brain tissue section which was subjected to the samerepeated imaging experiment. The decay profile of the ion signal withDESI analysis repetition is consistent with the accepted extractionmechanism.

FIG. 3. DESI-MS ion images obtained from a 15 μm thick mouse braincoronal section using DMF:EtOH (1:1) as the solvent system showing thedistribution of the ions at (A) m/z 834.3, PS(18:0/22:6); (B) m/z 888.6,ST(24:1); (C) m/z 890.7, ST(24:0); (D) m/z 885.6, PI(18:0/20:4) and (E)m/z 303.3, FA(20:4). Optical image of the same tissue section firstimaged by DESI-MS and then H&E stained is shown in (F). High qualitytwo-dimensional ion images were obtained under standard optimizedDESI-MS imaging conditions at a lateral resolution of approximately 180μm.

FIG. 4. DESI-MS imaging of human bladder cancerous and adjacent normaltissue sections using morphology friendly DMF:EtOH (1:1) as the solventsystem. Ion images show the distribution of (A) m/z 788.4,PS(18:0/18:1); (B) m/z 885.6, PI (18:0/20:4); (C) m/z 835.6,PI(16:0/18:1); (D) m/z 281.6, FA (18:1) and (E) m/z 537.2 (FA dimer).After the DESI-MS imaging experiment, the same tissue sections weresubjected to H&E staining (F), evaluated by expert pathologist, anddiagnosed as cancerous and normal. Representative mass spectra of (G)normal and (H) cancerous tissue are shown. Overlay of the ion image m/z537.2 and H&E stain of the same tissue section allowed a region ofnormal tissue to be detected within the cancerous tissue section (I).

FIG. 5. Non-destructive DESI-MS imaging allows immunohistochemistry tobe performed after imaging on the same tissue section when usingmorphologically friendly solvent systems. Optical images of the entiretissue and magnified brightfield optical images of H&E stained and p63IHC prostate tissue sections used are shown for (A) control samples, (B)samples imaged by DESI-MS using DMF:EtOH (1:1) as solvent system and (C)samples imaged by DESI-MS using standard ACN:H₂O (1:1) as solventsystem.

DETAILED DESCRIPTION

The present invention provides new methodologies that allow massspectrometry analysis of a lipid containing sample (e.g., DESI-MSimaging) to be performed in a non-destructive matter, so that otheranalyses of the same sample can be performed after the mass spectrometryanalysis is performed. A particular embodiment of the invention relatesto mass spectral tissue imaging using DESI. DESI-MS imaging has beenincreasingly applied in the biomedical field. Methods of the invention,which use a variety of solvent systems for imaging, allows DESI-MSimaging of chemical compounds to be performed on lipid containingsamples (e.g., tissue sections), while the morphology of the tissueremains unmodified. After DESI-MS imaging, the tissue can be used forH&E staining, immunohistochemistry, and any other tissue analysistechnique to obtain more information on the distribution of its chemicalconstituents.

A frozen mouse brain from a male mouse was purchased from RocklandImmunochemicals, Inc. (Gilbertsville, Pa., USA) and stored at −80° C.until it was sliced into coronary sections of varying thickness (2 μm, 3μm, 5 μm and 15 μm) using a Shandon SME Cryotome cryostat (GMI, Inc.,Ramsey, Minn., USA) and thaw mounted onto glass slides. The glass slideswere stored in a closed container at −80° C. until analysis, when theywere allowed to come to room temperature and dried in a dessicator forapproximately 15 minutes. All human tissue samples were handled inaccordance with approved institutional review board (IRB) protocols atIndiana University School of Medicine. Six human bladder cancer andpaired normal samples, four human prostate cancer and paired normalsamples and one human kidney cancer and paired normal sample wereobtained from the Indiana University Medical School Tissue Bank. Alltissue samples were flash frozen in liquid nitrogen at the time ofcollection and subsequently stored at −80° C. until sliced into 5 or 10μm thick sections. The 5 and 15 μm thick sections were used for DESI-MSimaging experiments followed by either p63 immunohistochemistry or H&Estain, respectively. Tissue sections not analyzed by DESI-MS were usedin control experiments. The thin tissue sections were thaw mounted toglass slides; each slide containing one section of tumor tissue and onesection of adjacent normal tissue from the same patient. The glassslides were stored in closed containers at −80° C. Prior to analysis,they were allowed to come to room temperature and then dried in adessicator for approximately 15 minutes.

The DESI ion source was a lab-built prototype, similar to a commercialsource from Prosolia Inc. (Indianapolis, Ind. USA), configured asdescribed elsewhere (Watrous J D, Alexandrov T, & Dorrestein PC (2011),Journal of Mass Spectrometry 46(2):209-222). It consists of an innercapillary (fused silica, 50 μm i.d., 150 μm o.d.) (PolymicroTechnologies, AZ, USA) for delivering the spray solvent and an outercapillary (250 μm i.d., 350 μm o.d.) for delivering nitrogen nebulizinggas. The DESI spray was positioned 2.5 mm from the tissue sample at anincident angle of 54°. A low collection angle of 10° was chosen toensure the most efficient collection of the material being desorbed. Thedistance between the spray and the inlet was 6.0 mm. Multiple spraysolvent systems were tested in the experiments, including ACN, H₂O,MeOH, ethanol (EtOH), tetrahydrofuran (THF), N,N-dimethylformamide(DMF), chloroform (CHCl₃), acetone and many of their binary mixtures ina ratio of (1:1). The only tertiary mixture investigated was ofACN:H₂O:DMF at different v/v proportions, such as (8:3:1 and 1:1:1).DESI-MS experiments were carried out in the negative ion mode, using a 5kV spray voltage and a flow rate of 0.5-1.5 μL/min depending on thesolvent system of choice. The nebulizing gas (N2) pressure was set forall experiments at 175-180 psi. The mass spectrometer used was a LTQlinear ion trap mass spectrometer controlled by XCalibur 2.0 software(Thermo Fisher Scientific, San Jose, Calif., USA).

Analysis were performed using an imaging approach. The tissues werescanned using a 2D moving stage in horizontal rows separated by a 150 μmvertical step for the mouse brain imaging assay (FIG. 3), and 250 μmvertical step for the human tissue imaging assays. For the DESI-MS assayshown in FIG. 2, the same mouse brain section was imaged 10 times withDMF:EtOH (1:1) and each analysis was performed in 10 lines of 250 μm.After one analysis was concluded, the moving stage was set to coordinate0,0 (x,y) for the new analysis to be performed. The surface moving stageincluded an XYZ integrated linear stage (Newport, Richmond, Calif., USA)and a rotary stage (Parker Automation, Irwin, Pa., USA). A softwareprogram allowed the conversion of the XCalibur 2.0 mass spectra files(.raw) into a format compatible with the Biomap software (freeware,http://www.maldi-msi.org). Spatially accurate images were assembledusing the BioMap software. The color scale is normalized to the mostintense (100% relative intensity) peak in the mass spectra.

Tissue sections were subjected to H&E staining after DESI-MS imaginganalysis or after being dried in a dessicator (control sections). Allchemicals used for the H&E staining were purchased from Sigma-Alrich(St. Louis, Mo., USA). The H&E staining was performed at roomtemperature: dip in MeOH for 2 minutes, rinse in water (10 dips), stainin Harris modified hematoxylin solution for 1.5 minutes, rinse in water(10 dips), 1 quick dip in 0.1% ammonia (blueing agent), rinse in water(10 dips), counterstain in Eosin Y (8 seconds), rinse in 100% EtOH (10dips), rinse again in 100% EtOH (10 dips), rinse in Xylene (6 dips) andrinse again in Xylene (6 dips). Sections were allowed to dry and coveredwith a glass cover slide. Immunohistochemistry assays were performed inthe Veterinary Department at Purdue University by Dr. Carol Bain, inaccordance to their standard protocol. The primary antibody p63(4A4):sc-8431 was purchased from Santa Cruz Biotechnology, INC (SantaCruz, Calif., USA).

Pathological evaluation of the human tissue sections that were eitherH&E stained or subjected to p63 immunohistochemistry was performed byDr. Liang Cheng, at IU School of Medicine in a blind fashion. Opticalimages of tissue sections were obtained using a SM-LUX BinocularBrightfield Microscope (Leitz, Wetzlar, Germany) under 16, 25 and 40×magnification.

The solvent system used in DESI tissue imaging is taught to be animportant technical parameter for optimization (Badu-Tawiah A, Bland C,Campbell D I, & Cooks R G (2010), Journal of the American Society forMass Spectrometry 21(4):572-579; and Green F M, Salter TL, Gilmore I S,Stokes P, & O'Connor G (2010), Analyst 135(4):731-737). Many studieshave shown that the chemical and physical properties of the solventsystem used affect the molecular information obtained during DESI-MStissue imaging (Ellis SR, et al. (2010), J. Am. Soc. Mass Spectrom.21(12):2095-2104). Optimization of the spray composition allows targetedclasses of compounds to be enhanced depending on the overall goal.Besides the chemical information, the effect of the solvent system onthe morphology of the tissue being analyzed is a factor in DESI-MSimaging. Commonly used DESI-MS imaging solvent systems, such as mixturesof water with methanol or acetonitrile (Wiseman J M, Ifa D R, Venter A,& Cooks R G (2008), Nature Protocols 3(3):517-524), with or without anacidic modifier, yield extensive chemical information but are known tocause depletion and destruction of the tissue sections, precluding anyconsecutive analysis to be performed. To overcome these problems,different solvents such as ACN, H₂O, MeOH, ethanol (EtOH),tetrahydrofuran (THF), N,N-dimethylformamide (DMF), chloroform (CHCl₃),acetone and mixtures of these were investigated in the analysis of 15 μmthick serial coronary mouse brain tissue sections.

A binary mixture of MEOH:H₂O (1:1, v/v) or ACN:H₂O (1:1, v/v) has beencommonly used in DESI imaging of brain tissue, yielding high signalintensity for polar lipids and free fatty acids (Eberlin L S, Ifa D R,Wu C, & Cooks R G (2010), Angewandte Chemie-International Edition49(5):873-876; and Wiseman J M, Ifa D R, Song Q Y, & Cooks R G (2006),Angewandte Chemie-International Edition 45(43):7188-7192). The majorityof the ions observed in the mass spectra obtained from the solventsystems tested here correspond to commonly observed lipid species inbrain tissue when using standard MeOH:H₂O (1:1), such as deprotonatedfree fatty acids, phosphatidylserines (PS), phosphatidylinositols (PI)and sulfatides (ST) (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010),Angewandte Chemie-International Edition 49(5):873-876). Variations inthe relative abundance of the lipid species and in the total ion signalobtained were observed depending on the solvent composition. Forinstance, spectra obtained when using pure methanol as the solventsystem showed higher relative abundance of fatty acid dimers in the m/z500-700 region of the mass spectrum. In particular, it was observed thatpure DMF yielded spectra with high total abundance and with chemicalinformation which is very similar to that which is obtained usingMeOH:H₂O.

Interestingly, the DMF spray was observed to not cause tissuedestruction. The effect of DMF in the tissue was further explored bycombining this solvent with other solvents in binary (1:1 v/v) andtertiary mixtures. The combination of DMF with either ACN, EtOH, THF orCHCl₃ yielded very high ion signal and chemical information similar towhat is seen using MeOH:H₂O. Combinations of DMF with either H₂O or MeOHgreatly enhanced the signal of low molecular weight compounds, such assmall metabolites, FAs and FA dimers (Eberlin L S, Ferreira C R, Dill AL, Ifa D R, & Cooks R G (2011) Desorption Electrospray Ionization MassSpectrometry for Lipid Characterization and Biological Tissue Imaging.Biochimica Et Biophysica Acta-Molecular And Cell Biology Of Lipidsaccepted). In terms of spray stability and total ion abundance, thecombinations of DMF with either EtOH or ACN are great solvent systemsfor tissue imaging experiments. The change in chemical informationobtained by DESI-MS using different solvent combinations can be comparedto the use of different matrices in MALDI imaging, but in DESI-MSimaging experiments, the “matrix” is delivered in real-time,spot-by-spot, without the need for sample preparation or without causingspatial delocalization of molecules.

Importantly, none of these solvent combinations were observed to causevisual damage to the 15 μm thick tissue sections that were analyzed.FIG. 1 shows the physical and chemical effect of two of the new solventsystems developed, DMF:EtOH (1:1) and DMF:H₂O (1:1) in comparison to thestandard MEOH:H₂O solvent system. DESI-MS conditions were kept identicalin all analyses performed. It is striking to observe that whileextensive chemical information was obtained from the tissue sectionswhen using DMF:H₂O and DMF:EtOH, tissue integrity was preserved.

As observed in the optical images shown of the DESI-MS experiment,damage to the tissue was insignificant when using DMF solvent systems.To confirm preservation of tissue integrity, H&E staining was performedon the tissue sections previously analyzed by DESI-MS. H&E staining is acommonly used histochemical protocol to evaluate cellular structure andtissue morphology by light microscopy. Careful microscopic examinationof the H&E tissue sections revealed no damage or change in the cellularmorphology of the sample after DESI analysis using DMF:EtOH and DMF:H₂Osolvent systems, while the tissue analyzed using MeOH:H₂O was found tobe altered and damaged, as was macroscopically observed. DESI-MSanalysis of sequential mouse brain tissue sections of 2, 3 and 5 μmthicknesses was also performed, and sequential H&E staining of thetissue sections also revealed that no morphological damage occurredfollowing DESI-MS analysis using DMF:EtOH or DMF:ACN as the solventsystem.

The physical and chemical effect of the DMF:EtOH solvent system wasfurther investigated by performing several DESI-MS analyses of the samemouse brain tissue section. The same tissue region of a 5 μm and a 15 μmthick tissue section were analyzed 10 times using the DESI-MS movingstage system. Mass spectra were recorded for 10 rows (250 μm step size)of each mouse brain section and after 10 analyses had been performed,each tissue section was H&E stained and observed under brightfieldmicroscopy under 16-40× magnification. FIG. 2A and B show the massspectra obtained from the gray matter region of the 5 μm thick mousebrain tissue section from the 5^(th) row scanned using DMF:EtOH in the1^(st) and 10^(th) DESI-MS analysis, respectively. The ion count isapproximately 60 times greater in the 1^(st) analysis of the mouse braincompared to the ion count obtained in the 10^(th) analysis of the sameregion. The ion count of the main ion observed in the gray matter regionof the 5^(th) row scanned, m/z 834.4 (PS 18:0/22:6), was plotted as afunction of the DESI-MS analysis number for both the 5 μm and a 15 μmthick tissue sections, shown in FIG. 2C.

Interestingly, the signal of the typical ion of m/z 834.4 obtained inthe 3^(rd) or even 4^(th) DESI-MS analysis is still observed at highintensities. Furthermore, the decay profile of the ion count isconsistent with the extraction mechanism proposed for DESI-MS (Costa A B& Cooks R G (2008), Chem. Phys. Lett. 464(1-3):1-8). While a MeOH:H₂Ospray extracts the chemical compounds from the tissue cells resulting intissue damage, the DMF based solvent system is able to extract thechemical compounds from the tissue section without disturbing the tissuemorphology. H&E staining of both a 5 μm and a 15 μm thick tissue sectionafter ten DESI-MS imaging analyses revealed no damage to the tissue,indicating that the repetitive removal of the phospholipids by the DESIsolvent spray does not affect the morphology of the cells. In fact, theextraction process that occurs in DESI-MS is comparable to the fixativeprocedures commonly used in histology for lipid removal (DiDonato D &Brasaemle DL (2003), Journal of Histochemistry & Cytochemistry51(6):773-780), such as the alcohol wash used in the initial step of theH&E staining data. This alcohol wash step extracts the majority ofcellular phospholipids while the cellular cytoskeletal elements are keptintact. Since hematoxylin stains nucleoproteins and eosin stainsintracellular and extracellular proteins, the removal of the lipidcontent with conservation of the tissue integrity by DESI-MS should notinterfere with this standard histochemistry protocol. Importantly, theuse of DMF based solvent systems or even other solvent systems withsimilar morphologically-friendly properties allows pathologicalevaluation to be performed on the same tissue section previouslyanalyzed by DESI-MS but with acquisition of complementary results.

All combinations of DMF with other solvents used in the DESI-MS assayson mouse brain tissue sections were found to not destroy the nativemorphology of the tissue. Other pure solvents, such as ACN, DMF, THF,ethanol and others did not cause damage to the tissue integrity asobserved in the H&E stains. A few other combinations that did notcontain DMF, such as ACN:EtOH (1:1), MeOH:CHCl₃ (1:1) and ACN:CHCl₃(1:1), did not destroy the native morphology of the tissue. Themorphological effect that the DESI spray has on tissue appear to berelated to the physical and chemical properties of the solvent systemsitself.

While solubility of the proteic cellular and extracellular components ofthe tissue section in the DESI spray solvent system plays a role in theconservation of the tissue morphology integrity, the physical propertiesof the solvent system such as surface tension and its effects on thedynamics of the DESI spray primary droplets also impact the damagecaused to the tissue. When solubilization of cellular and extracellularcomponents that keep cellular morphological integrity intact occurs, thetissue becomes more susceptible to the mechanical action of the DESIspray droplets. Therefore, tissue damage should be related to bothsolubilization of tissue components and mechanical action of the DESIspray system. The fact that the morphologically-friendly solvent systemsdescribed here do not disturb tissue integrity appear to be related tothe physical properties of the DESI spray primary droplets, but also onthe solubility of tissue components on the solvent system.

Chemical information and image quality are important factors in DESI-MSimaging applications. The geometric parameters of the DESI spray as wellas the choice of solvent system, gas pressure and solvent flow areimportant when optimizing imaging conditions. When the solvent system ismodified, it is important to observe that the spray spot is stable andthat the ion signal intensity is maximized for obtaining good quality 2Dchemical images.

FIG. 3 shows ion images of a mouse brain tissue section obtained usingDMF:EtOH as the solvent system. The spray geometry and gas pressureconditions used in this imaging experiment are standard for DESI-MSimaging applications (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010),Angewandte Chemie-International Edition 49(5):873-876; and Ifa D R,Wiseman J M, Song Q Y, & Cooks R G (2007), International Journal of MassSpectrometry 259(1-3):8-15). In terms of solvent flow, it was observedthat at a regular DESI-MS imaging flow rate of 1.5 μL/min a larger spotsize is obtained with DMF solvent combinations as compared to standardmixtures of water with MEOH or ACN at the same flow. The high stabilityand larger diameter of the spray spot can be associated with the higherboiling point of DMF (153° C.), when compared to the boiling point ofsolvents as MeOH and ACN. Smaller diameter spray spots can be achievedby using either a lower solvent flow rate or by mixing DMF with a higherratio of a solvent with higher volatility, such as a mixture of DMF:EtOH(1:2). A solvent flow of 0.5 μL/min was used for the mouse brain imagingexperiments using binary mixtures of DMF so that a spot size ofapproximately 180 μm was obtained. Lower solvent consumption as a resultof using a lower flow rate is advantageous in DESI-MS imagingapplications.

In the images shown in FIG. 3, two distinctive MS peak patternsassociated with the lipid compositions representative of the gray andwhite matter of the brain were observed in the negative-ion mode for thebrain section analyzed (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010),Angewandte Chemie-International Edition 49(5):873-876). The ion imagesof m/z 834.3, PS 18:0/22:6 (FIG. 3A), m/z 885.6, PI 18:0/20:4 (FIG. 3D)and m/z 303.3,FA 20:4 (FIG. 3E) show a homogeneous distribution in thebrain gray matter, which are complementary and distinct from the ionimages of m/z 888.8 (ST 24:1, FIG. 3B) and m/z 890.7 (ST 24:0, FIG. 3C),which are homogeneously distributed in the mouse brain white matter.FIG. 3F shows the optical image of the same tissue section which was H&Estained after DESI-MS imaging was performed. This order of analysis inwhich ambient MS imaging is performed followed by histochemical analysisof the same tissue section is comparable to the “post-acquisitionstaining” methodology used in MALDI-MS imaging (Schwamborn K, et al.(2007), International Journal of Molecular Medicine 20(2):155-159). Thehigh-quality 2D DESI-MS ion images can be directly compared and evenoverlaid with the H&E stained tissue section, allowing a bettercorrelation between the spatial distribution of the lipid speciesdetected and the substructures of the mouse brain.

The capability to perform DESI-MS imaging and histochemical analysis ofthe same tissue section is important in the investigation of diseasedtissue. The comparison of histological features from stained sectionswith corresponding molecular images obtained by ambient imaging MS isimportant for accurate correlations between molecular signatures andtissue disease state. This is especially true in the analysis ofcancerous tissue sections which are very often highly heterogeneous,with regions of containing various tumor cell concentrations (Agar N YR, et al. (2011), Neurosurgery 68(2):280-290), infiltrative normaltissue (Dill A L, et al. (2011), Chemistry-a European Journal17(10):2897-2902), precancerous lesions (Eberlin LS, et al. (2010),Analytical Chemistry 82(9):3430-3434), etc. Integration of DESI-MSimaging into a traditional histopathology workflow required that themass spectrometric analysis not interfere with the morphology of thetissue section. Provided this is the case, the combination of the twodifferent types of data (as represented by the case of superimposedimages) greatly increases discrimination between different tissue typesincluding that between diseased and healthy tissue.

To investigate this capability, human bladder, kidney and prostatecancer tissues along with adjacent normal samples were analyzed byDESI-MS imaging in the negative ion mode using one of our histologycompatible solvent system and sequentially H&E stained. The lipidspecies present in the tissue sections were identified based oncollision-induced dissociation (CID) tandem MS experiments andcomparison of the generated product ion spectra with literature data(Hsu F F & Turk J (2000), Journal of the American Society for MassSpectrometry 11(11):986-999). FIG. 4 shows a series of negative ion modeDESI-MS ion images of species commonly observed in human bladdertransitional cell carcinoma and adjacent normal tissue from sampleUH0210-13. FIGS. 4A, B, C, D and E show the ion images obtained for theions at m/z 788.4 (PS(18:0/18:1)), m/z 885.6 (PI(18:0/20:4)), m/z 835.6(PI(16:0/18:1)), m/z 281.6 (FA 18:1) and m/z 537.2 (FA dimer).

As previously reported for DESI-MS imaging of human bladder cancer incombination with statistical analysis using a standard ACN:H₂O (1:1)solvent system, the ions that most significantly contribute to thediscrimination between cancerous and normal bladder tissue are the freefatty acid and the fatty acid dimers, which consistently appear atincreased intensities in the ion images of cancerous tissue whencompared to normal tissue using the morphology friendly solvent system,DMF:EtOH (1:1) (FIGS. 4C and D; Dill A L, et al. (2011), Chemistry-aEuropean Journal 17(10):2897-2902; and Dill A L, et al. (2009),Analytical Chemistry 81(21):8758-8764). Representative mass spectraobtained for the cancerous and normal tissue sections are shown in FIGS.4G and H. Many other individual ions observed in the mass spectra werefound at different intensities in the normal and cancerous tissues asobserved in extracted DESI-MS ion images.

The optical image of the same tissue sections stained with H&E afterDESI-MS imaging analysis is shown in FIG. 4F and were used to obtain ahistopathological diagnosis. Detailed pathological examination of theH&E stained sections confirmed that there was no morphological damage tothe tissue sections as a result of DESI-MS imaging analysis, allowing astraightforward diagnosis of the sections as cancerous and normal. Nodifference in cell morphology or tissue integrity was observed at themicroscopic scale when the H&E stained tissue section of the DESI-MSimaged tissue was compared to a control tissue section.

The non-destructive nature of the DMF based solvent system enables ionimages to be overlaid with the H&E stain of the same tissue section forunambiguous diagnosis and correlation. For example, a small region oftissue within the cancerous section detected by DESI-MS as negative forbladder cancer based on the distribution of the FA dimer m/z 537.2 wasconfirmed as normal tissue by pathological evaluation of the overlaidDESI-MS ion image and H&E stain of the same tissue section. Thisunambiguous correlation is made possible through the use of themorphologically friendly solvent systems so that the histological datacan be considered in combination with the DESI-MS imaging data. H&Estained serial sections of the same sample imaged using standard ACN:H₂O(1:1) revealed that the tissue integrity was completely destroyed andwere inadequate for pathological evaluation. The same histologicalobservation that DESI imaging is histology compatible was obtained inthe analysis of the H&E stained sections of five other human bladdercancer and paired normal samples, four human prostate cancer and pairednormal samples and one kidney cancer and paired normal sample initiallyimaged by DESI-MS with a morphologically friendly solvent system.Previously reported molecular information that allowed a diagnosis to beobtained for these types of cancer was consistent using the new solventsystem. The capability of DESI-MS imaging to be histology compatible wasfurther investigated by performing immunohistochemical (IHC) analysiswith p63 antibody on bladder and prostate cancer tissue sections, whichwas performed after DESI-MS imaging. The gene p63 is one of the mostcommonly used basal cell-specific markers in the diagnosis of prostatecancer, whose expression is known to be down-regulated in adenocarcinomaof the prostate when compared to normal prostate tissue (Signoretti S,et al. (2000), American Journal of Pathology 157(6):1769-1775). NegativeIHC staining of tumor protein p63 is commonly used as a clinical toolfor identifying prostate cancerous tissue. The role of p63 in bladdercarcinogenesis is not as clear as in prostate cancer (Comperat E, et al.(2006), Virchows Archiv 448(3):319-324), and positive staining of p63 istypically associated with both benign and malignant bladder epithelialcells.

Two bladder cancer samples and two prostate cancer samples weresubjected to p63 IHC after DESI-MS imaging on the same tissue section.Detailed pathological evaluation of the tissue sections that weresubjected to IHC after DESI-MS imaging confirmed that the DESI-MSanalysis of the tissue lipid content did not interfere with the p63 IHCprotocol, as the tissue remained intact after the imaging experiment.p63 IHC of the bladder sample was found to be positive for bothcancerous and normal tissue sections. For the prostate cancer sample,UH0002-20, it was subjected to p63 IHC after DESI-MS imaging and againno damage to the morphology of the tissue was observed (FIG. 5),allowing a diagnosis of cancerous and adjacent normal tissue to beachieved, which correlated with the cholesterol sulfate signalpreviously reported as a possible prostate cancer biomarker usingDESI-MS imaging (Eberlin LS, et al. (2010), Analytical Chemistry82(9):3430-3434.). Also, these findings confirmed that protein positionin the tissue samples remained unchanged, which is probably due to theinsolubility of these proteins in the solvent combinations used.

The results reported here introduce a novel capability of histologicallycompatible ambient molecular imaging by DESI-MS. The feasibility ofDESI-MS imaging to be performed while tissue integrity and cellmorphology is conserved allows ambient mass spectrometric analysis oftissue to be combined with traditional histopathology with the goal ofproviding better disease diagnostics. As DESI-MS imaging usinghistologically friendly solvent systems does not interfere withpathological analysis, the technique could be included as the initialstep in the clinical tissue analysis workflow.

Methods reported herein will allow DESI-MS to be more broadly applied inthe biomedical field, such as in intraoperative applications. Inadditional to biomedical applications, the morphologically compatiblesolvent system allows DESI-MS imaging to be combined to other analyticaltechniques for chemical analysis of the same tissue section.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

What is claimed is:
 1. A method for diagnosing an abnormality, themethod comprising: performing a mass spectral analysis of a lipidcontaining tissue sample by directing a liquid phase that does notdestroy native tissue morphology from an ionization probe onto the lipidcontaining tissue sample to thereby desorb one or more lipids from thesample that are analyzed by a mass spectrometer; producing a massspectral image of the lipid containing tissue sample based on results ofthe mass spectral analysis; performing a histochemistry analysistechnique on said sample to thereby stain the sample; producing anoptical image of the stained sample; overlaying the mass spectral imagewith the optical image of the stained sample to produce an overlaidimage; and detecting an abnormality based on an analysis of the overlaidimage.
 2. The method of claim 1 wherein the liquid phase is a solventcomprising dimethylformamide (DMF), acetonitrile (ACN), ortetrahydrofuran (THF).
 3. The method according to claim 2, wherein theliquid phase comprises at least one other component.
 4. The methodaccording to claim 3, wherein the at least one other component isselected from the group consisting of ethanol (EtOH), water (H₂O),acetonitrile (ACN), and a combination thereof.
 5. The method accordingto claim 1, wherein the liquid phase is selected from the groupconsisting of: acetonitrile (ACN):ethanol (EtOH); methanol(MeOH):chloroform (CHCl₃); and acetonitrile (ACN):chloroform (CHCl₃). 6.The method according to claim 1, wherein the histochemistry analysistechnique is H&E staining.